Once all reactions are complete, transfer the tubes to a microfuge and spin for 10 minutes at full speed.

Gently remove tubes from centrifuge.

Measure the absorbance at 420nm and 550nm. (Use UV-Vis protocol on Nanodrop).

Calculate Miller Units as:

or

where:

Abs420 is the absorbance of the yellow o-nitrophenol,

Abs550 is the scatter from cell debris, which, when multiplied by 1.75 approximates the scatter observed at 420nm,

t = reaction time in minutes,

v = volume of culture assayed in milliliters,

Abs600† reflects cell density.

Comments on the assay

Reshma 11:28, 15 October 2007 (CDT): Miller recommends a culture with OD600 = 0.28 to 0.70. However, he claims that overnight cultures can also be used but that exponentially growing cells give more precise assays [1].

When is the reaction done? I never found a good answer for this in the literature. If I let a reaction go to completion, I measured an Abs420 of ~2-3. Of course, this is out of the reliable range of the spectrophotometer, but it gives an indication of how far the reaction can go. I got reproducible data when the yellow color was just detectable before adding the stop solution up to about the color of LB broth before stopping. Remember, you need the substrate to saturate the enzyme during the course of the reaction, so don't let them go too far. I routinely make three separate measurements for each culture and average them.